Abstract

The use of a DNA polymerise theta (PolQ) enzyme in a method of template-independent nucleic acid synthesis. The method comprises providing an initiator sequence, adding a 3'-blocked nucleotide triphosphate to the initiator in the presence of PolQ, removal of all reagents from the initiator, cleaving the blocking group from the 3'-blocked nucleotide triphosphate and removal of the cleaving agent. The 3'-block may be 3'-O-azidomethyl, 3'-aminoxy or 3'-O-allyl group.

Description

NOVEL USE

FIELD OF THE INVENTION

The invention relates to the use of a DNA polymerase theta (PolQ) enzyme in a method of template-independent nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis.

BACKGROUND OF THE INVENTION

Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community’s ability to artificially synthesise DNA, RNA and proteins.

Artificial DNA synthesis - a £1 billion and growing market - allows biotechnology and pharmaceutical companies to develop a range of peptide therapeutics, such as insulin for the treatment of diabetes. It allows researchers to characterise cellular proteins to develop new small molecule therapies for the treatment of diseases our ageing population faces today, such as heart disease and cancer. It even paves the way forward to creating life, as the Venter Institute demonstrated in 2010 when they placed an artificially synthesised genome into a bacterial cell.

However, current DNA synthesis technology does not meet the demands of the biotechnology industry. While the benefits of DNA synthesis are numerous, an oft-mentioned problem prevents the further growth of the artificial DNA synthesis industry, and thus the biotechnology field. Despite being a mature technology, it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length, and most DNA synthesis companies only offer up to 120 nucleotides. In comparison, an average protein-coding gene is of the order of 2000 - 3000 nucleotides, and an average eukaryotic genome numbers in the billions of nucleotides. Thus, all major gene synthesis companies today rely on variations of a ‘synthesise and stitch’ technique, where overlapping 40-60-mer fragments are synthesised and stitched together by PCR (see Young, L. et al. (2004) Nucleic Acid Res. 32, e59). Current methods offered by the gene synthesis industry generally allow up to 3 kb in length for routine production.

The reason DNA cannot be synthesised beyond 120-200 nucleotides at a time is due to the current methodology for generating DNA, which uses synthetic chemistry (/.e., phosphoramidite technology) to couple a nucleotide one at a time to make DNA. As the efficiency of each nucleotide-coupling step is 95.0 - 99.5% efficient, it is mathematically impossible to synthesise DNA longer than 200 nucleotides in acceptable yields. The Venter Institute illustrated this laborious process by spending 4 years and 20 million USD to synthesise the relatively small genome of a bacterium (see Gibson, D. G. et al. (2010) Science 329, 52-56).

Known methods of DNA sequencing use template-dependent DNA polymerases to add 3’-reversibly terminated nucleotides to a growing double-stranded substrate (see, Bentley, D. R. etal. (2008) Nature 456, 53-59). In the ‘sequencing-by-synthesis’ process, each added nucleotide contains a dye, allowing the user to identify the exact sequence of the template strand. Albeit on double-stranded DNA, this technology is able to produce strands of between 500-1000 bps long. However, this technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template.

Various attempts have been made to use an enzyme called terminal deoxynucleotidyl transferase (TdT) for controlled de novo single-stranded DNA synthesis (see Ud-Dean, S. M. M. (2009) Syst Synth Boil 2, 67-73, US 5,763,594 and US 8,808,989). Uncontrolled de novo single-stranded DNA synthesis, as opposed to controlled, takes advantage of TdT’s deoxynucleotide triphosphate (dNTP) 3’ tailing properties on single-stranded DNA to create, for example, homopolymeric adaptor sequences for next-generation sequencing library preparation (see Roychoudhury R., etal. (1976) Nucleic Acids Res 3, 101—116 and WO 2003/050242). A reversible deoxynucleotide triphosphate termination technology needs to be employed to prevent uncontrolled addition of dNTPs to the 3’-end of a growing DNA strand. The development of a controlled single-stranded DNA synthesis process through TdT would be invaluable to in situ DNA synthesis for gene assembly or hybridization microarrays as it removes the need for an anhydrous environment and allows the use of various polymers incompatible with organic solvents (see Blanchard, A. P. (1996) Biosens Bioelectron 11, 687-690 and US 7,534,561).

However, TdT displays differential incorporation efficiency depending on the identity of the nitrogenous base of the incoming 3'-0-reversibly terminated nucleotide 5'-triphosphate. Indeed, TdT incorporation efficiency of guanosine, cytidine, and thymidine is significantly higher compared with adenosine. In order for enzymatic de novo DNA synthesis to be practically useful in creating biologically relevant sequences, all four bases must be incorporated with high efficiency.

Whereas TdT displays high incorporation efficiency with all canonical bases other than adenosine, DNA polymerase theta (PolQ) is the exact opposite. PolQ, a terminal transferase like TdT, displays highest incorporation efficiencies on unblocked adenosine 5'-triphosphates (see Kent, T. etal., (2016) eLife 5, e13740). Due to the base selectivity of each of the known terminal transferases, one can design a process where TdT is used to incorporate reversibly blocked guanosine, thymidine, and cytidine 5'-triphosphates, whereas PolQ is used to incorporate reversibly blocked adenosine 5'-triphosphates.

There is therefore a need to identify enzymes that readily incorporate 3’-0 reversibly terminated adenosine triphosphates and modified said enzyme to incorporate 3’-0 reversibly terminated adenosine triphosphates in a fashion useful for biotechnology and single-stranded DNA synthesis processes in order to provide an improved method of nucleic acid synthesis that is able to overcome the problems associated with currently available methods.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided the use of a DNA polymerase theta (PolQ) enzyme in a method of template-independent nucleic acid synthesis (Figure 1).

According to a second aspect of the invention, there is provided a method of nucleic acid synthesis, which comprises the steps of: (a) providing an initiator sequence; (b) adding a 3’-blocked nucleotide triphosphate to said initiator sequence in the presence of PolQ as defined in the first aspect of the invention; (c) removal of all reagents from the initiator sequence; (d) cleaving the blocking group from the 3’-blocked nucleotide triphosphate in the presence of a cleaving agent; (e) removal of the cleaving agent.

According to a further aspect of the invention, there is provided the use of a kit in a method of template-independent nucleic acid synthesis, wherein said kit comprises a PolQ as defined in the first aspect of the invention optionally in combination with one or more components selected from: an initiator sequence, one or more 3’-blocked nucleotide triphosphates, inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae, and a cleaving agent; further optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematic of enzymatic DNA synthesis process. Starting from the top of the diagram, an immobilised strand of DNA with a deprotected 3’-end is exposed to an extension mixture composed of PolQ or TdT, a base-specific 3’-blocked nucleotide triphosphate, inorganic pyrophosphatase to reduce the buildup of inorganic pyrophosphate, and appropriate buffers/salts for optimal enzyme activity and stability. The protein adds one protected nucleotide to the immobilised DNA strand (bottom of diagram). The extension mixture is then removed with wash mixture and optionally recycled. The immobilised (n+1) DNA strand is then washed with a cleavage mixture to cleave the 3’-protecting group, enabling reaction in the next cycle. In the cleavage mixture, denaturant may be present to disrupt any secondary structures. During this step, the temperature may be raised to assist in cleavage and disruption of secondary structures. The immobilised DNA is treated with wash mixture to remove leftover cleavage mixture. Steps 1-4 may be repeated with an appropriate nucleotide triphosphate until the desired oligonucleotide sequence is achieved.

Figure 3: Simplified schematic representation of a column-based flow instrument used in DNA synthesis. A computer (302) controls two pumps and a solution mixing chamber (311). Pump 1 (304) selectively pumps extension solution (301), wash solution (305) or cleavage solution (310) into the mixing chamber. Pump 2 (306) selectively pumps a single 3’-blocked nucleotide triphosphate (TP) solution containing either 3’-blocked A(adenine)TP (303), T(thymine)TP (307), G(guanine)TP (308), or C(cytosine)TP (309) into the chamber. The computer controlled mixing chamber then passes appropriate solution ratios from pump 1 and pump 2 into a column based DNA synthesis chamber (312). A heating element (313) ensures that the DNA synthesis column remains at the necessary temperature for the synthesis process to take place. Upon exiting the DNA synthesis chamber, the reaction solution either enters a recycling vessel (314) for future use, a waste vessel (316) or moves on to a polymerase chain reaction (PCR) step (315) for amplification of the resultant DNA. PCR completion leads to the final product (317).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided the use of a DNA polymerase theta (PolQ) enzyme in a method of template-independent nucleic acid synthesis.

References herein to the term “PolQ” refer to a DNA polymerase theta enzyme comprising SEQ ID NO: 1, functional fragments or homologues thereof, and purified and recombinant forms of said enzyme. PolQ is also commonly known as ΡοΙΘ and any such terms may be used interchangeably. PolQ is a translesion polymerase belonging to the A-family of DNA polymerases. PolQ is specifically implicated in repairing double-stranded DNA breaks in a highly error-prone mechanism called alternative end-joining (otherwise known as microhomology-mediated end-joining) as opposed to the better understood repair mechanisms, which are homologous recombination and non-homologous end-joining (see Villanueva etal., (2015) Nat Rev Cancer 15, 136 and van Schendel etal., (2015) Nat Commun 6, 7394). PolQ as a result was identified as a drug target for the purpose of suppressing of tumour growth (see Mateos-Gomez et al., (2015) Nature 518, 254-257).

References herein to a “method of template-independent nucleic acid synthesis” refer to a method of nucleic acid synthesis which does not require a template DNA/RNA strand, i.e. the nucleic acid can be synthesised de novo. In one embodiment, the nucleic acid is DNA. In an alternative embodiment, the nucleic acid is RNA.

In one embodiment, the DNA polymerase theta (PolQ) comprises an amino acid of sequence:

Since commercially available or mutant recombinant TdT does not readily incorporate 3’-O reversibly terminated nucleotides, specifically dATP, a terminal transferase capable of high-yielding 2'-deoxyadenosine 5'-triphosphate (dATP) incorporation is desirable in an enzymatic-based de novo nucleic acid synthesis process. PolQ was shown to have intrinsic terminal transferase abilities in vivo (see Mateos-Gomez et al., (2015) Nature 518, 254-257) and in vitro (see Kent, T. etal., (2016) eLife 5, e13740). Here, the inventors surprisingly show that PolQ can be adapted to perform a method of template-independent nucleic acid synthesis. As such, the present invention relates to the identification of PolQ enzymes for the incorporation of 3'-blocked nucleotide triphosphates, such as 3'-azidomethyl 2'-deoxyadenosine 5'-triphosphate (dATP), in template-independent, enzymatic-based de novo nucleic acid synthesis.

Furthermore, the present invention demonstrates PolQ is an effective, complementary terminal transferase to TdT for the incorporation of 3'-blocked dATP in a method of template-independent nucleic acid synthesis.

Furthermore, the present invention relates to engineered, non-natural PolQ enzymes for increased incorporation of nucleotide triphosphates containing 3’-0 reversibly terminating moieties to be useful for the controlled de novo synthesis of single-stranded DNA.

The use described herein has significant advantages, such as the ability to rapidly produce long lengths of DNA while still maintaining high yields and without using any toxic organic solvents.

References herein to a “method of nucleic acid synthesis” include methods of synthesising lengths of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) wherein a strand of nucleic acid (n) is extended by adding a further nucleotide (n+1). In one embodiment, the nucleic acid is DNA. In an alternative embodiment, the nucleic acid is RNA.

References herein to a “method of DNA synthesis” refer to a method of DNA strand synthesis wherein a DNA strand (n) is extended by adding a further nucleotide (n+1). The method described herein provides a novel use of PolQ of the invention and 3’-reversibly blocked nucleotide triphosphates to sequentially add nucleotides in de novo DNA strand synthesis which has several advantages over the DNA synthesis methods currently known in the art.

Current synthetic methods for coupling nucleotides to form sequence-specific DNA have reached asymptotic length limits, therefore a new method of de novo DNA synthesis is required. Synthetic DNA synthesis methods also have the disadvantage of using toxic organic solvents and additives (e.g., acetonitrile, acetic anhydride, trichloroacetic acid, pyridine, etc.), which are harmful to the environment.

An alternative, enzymatic method of nucleic acid synthesis is desirable. Natural enzymes such as DNA polymerases are able to add 50,000 nucleotides before disassociation. However, DNA polymerases require a template strand, thereby defeating the purpose of de novo strand synthesis.

This enzymatic approach means that the method herein disclosed, has the particular advantage of being able to produce DNA strands beyond the 120-200 nucleotide limit of current synthetic DNA synthesis methods. Furthermore, this enzymatic method avoids the need to use strong organic solvents which may be harmful to the environment.

It will be understood that the term ‘functional equivalent’ refers to polypeptides which have different polypeptide sequences, but can perform the same function, i.e., catalyse the addition of a nucleotide triphosphate onto the 3’-end of a DNA strand in a template-dependent manner.

In one embodiment, PolQ is a non-natural derivative of PolQ, such as a functional fragment or homolog of PolQ.

It will be appreciated that references herein to “identity” and “sequence identity” are to be understood as meaning the percentage identity between two protein sequences, e.g., SEQ ID NO: X and NO: Y, which is the sum of the common amino acids between aligned sequences SEQ ID NO: X and SEQ ID NO: Y, divided by the shorter length of either SEQ ID NO: X or SEQ ID NO: Y, expressed as a percentage.

According to a second aspect of the invention, there is provided a method of nucleic acid synthesis, which comprises the steps of: (a) providing an initiator sequence; (b) adding a 3’-reversibly terminated nucleotide triphosphate to said initiator sequence in the presence of PolQ as defined in the first aspect of the invention; (c) removal of all reagents from the initiator sequence; (d) cleaving the blocking group from the 3’-blocked nucleotide triphosphate in the presence of a cleaving agent; (e) removal of the cleaving agent.

In one embodiment, step (c) comprises removal of nucleotide triphosphates and PolQ. Thus, according to one particular aspect of the invention, there is provided a method of nucleic acid synthesis, which comprises the steps of: (a) providing an initiator sequence; (b) adding a 3’-reversibly terminated nucleotide triphosphate to said initiator sequence in the presence of a PolQ as defined in the first aspect of the invention; (c) removal of nucleotide triphosphates and PolQ; (d) cleaving the blocking group from the 3’-blocked nucleotide triphosphate in the presence of a cleaving agent; (e) removal of the cleaving agent.

In another embodiment, a terminal deoxynucleotidyl transferase (TdT) is used to add 3'-blocked thymidine 5'-triphosphate, cytidine 5'-triphosphate, and guanosine 5'-triphosphate whereas PolQ is used to add 3'-blocked adenosine 5'-triphosphate. Thus, according to one particular aspect of the invention, there is provided a method of nucleic acid synthesis, which comprises the steps of: (a) providing an initiator sequence; (b) adding either a 3’-blocked thymidine 5'-triphosphate, cytidine 5'-triphosphate, or guanosine 5'-triphosphate to said initiator sequence in the presence of a TdT or adding a 3’-blocked adenosine 5'-triphosphate to said initiator sequence in the presence of a PolQ enzyme as defined in the first aspect of the invention; (c) removal of nucleotide triphosphates and TdT or PolQ; (d) cleaving the blocking group from the 3’-blocked nucleotide triphosphate in the presence of a cleaving agent; (e) removal of the cleaving agent.

It will be understood that steps (b) to (e) of the method may be repeated multiple times to produce a DNA or RNA strand of a desired length. Therefore, in one embodiment, greater than 1 nucleotide is added to the initiator sequence, such as greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides are added to the initiator sequence by repeating steps (b) to (e). In a further embodiment, greater than 200 nucleotides are added, such as greater than 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides. 3’-blocked nucleotide triphosphates

Therefore, references herein to ‘3’-blocked nucleotide triphosphates’ refer to nucleotide triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the 3’ end which prevents further addition of nucleotides, i.e., by replacing the 3’-OH group with a protecting group.

It will be understood that references herein to ‘3’-block’, ‘3’-blocking group’ or‘3’-protecting group’ refer to the group attached to the 3’ end of the nucleotide triphosphate which prevents further nucleotide addition. The present method uses reversible 3’-blocking groups which can be removed by cleavage to allow the addition of further nucleotides. By contrast, irreversible 3’-blocking groups refer to dNTPs where the 3’-OH group can neither be exposed nor uncovered by cleavage.

There exist several documented reversible protecting groups, such as azidomethyl, aminoxy, and allyl, which can be applied to the method described herein. Examples of suitable protecting groups are described in Greene's Protective Groups in Organic Synthesis, (Wuts, P.G.M. &amp; Greene, T.W. (2012) 4th Ed., John Wiley &amp; Sons).

In one embodiment, the 3’-blocked nucleotide triphosphate is blocked by a reversible protecting group. In an alternative embodiment, the 3’-blocked nucleotide triphosphate is blocked by an irreversible protecting group.

Therefore, in one embodiment, the 3’-blocked nucleotide triphosphate is blocked by either a 3’-0-methyl, 3’-azido, 3’-0-azidomethyl, 3’-aminoxy or 3’-0-allyl group. In a further embodiment, the 3’-blocked nucleotide triphosphate is blocked by either a 3’-0-azidomethyl, 3’-aminoxy or 3’-0-allyl group. In an alternative embodiment, the 3’-blocked nucleotide triphosphate is blocked by either a 3’-0-methyl or 3’-azido group.

In one embodiment, the 3’-blocked nucleotide triphosphate is selected from:

wherein ‘X’ is as defined hereinbefore.

It will be understood that ‘PPP’ in the structures shown herein represents a triphosphate group.

Cleaving agent

References herein to ‘cleaving agent’ refer to a substance which is able to cleave the 3’-blocking group from the 3’-blocked nucleotide triphosphate.

In one embodiment, the cleaving agent is a chemical cleaving agent. In an alternative embodiment, the cleaving agent is an enzymatic cleaving agent.

It will be understood by the person skilled in the art that the selection of a cleaving agent is dependent on the type of 3’-nucleotide blocking group used. For instance, tris(2-carboxyethyl)phosphine (TCEP) or ruthenium complexes can be used to cleave a 3’-0-azidomethyl group, palladium complexes can be used to cleave a 3’-0-allyl group, or sodium nitrite can be used to cleave a 3’-aminoxy group. Therefore, in one embodiment, the cleaving agent is selected from: tris(2-carboxyethyl)phosphine (TCEP); tris(bipyridine)ruthenium (II) chloride and sodium ascorbate; a palladium complex; or sodium nitrite.

In one embodiment, the cleaving agent is added in the presence of a cleavage solution comprising a denaturant, such as urea, guanidinium chloride, formamide or betaine. The addition of a denaturant has the advantage of being able to disrupt any undesirable secondary structures in the DNA. In a further embodiment, the cleavage solution comprises one or more buffers. It will be understood by the person skilled in the art that the choice of buffer is dependent on the exact cleavage chemistry and cleaving agent required.

Initiator Sequences

References herein to an ‘initiator sequence’ refer to a short oligonucleotide with a free 3’-end which the 3’-blocked nucleotide triphosphate can be attached to. In one embodiment, the initiator sequence is a DNA initiator sequence. In an alternative embodiment, the initiator sequence is an RNA initiator sequence.

References herein to a ‘DNA initiator sequence’ refer to a small sequence of DNA which the 3’-blocked nucleotide triphosphate can be attached to, i.e. DNA will be synthesised from the end of the DNA initiator sequence.

In one embodiment, the initiator sequence is between 5 and 50 nucleotides long, such as between 5 and 30 nucleotides long (i.e. between 10 and 30), in particular between 5 and 20 nucleotides long (i.e., approximately 20 nucleotides long), more particularly 5 to 15 nucleotides long, for example 10 to 15 nucleotides long, especially 12 nucleotides long.

In one embodiment, the initiator sequence has the following sequence: 5’-CGTTAACATATT-3’ (SEQ ID NO: 2).

In one embodiment, the initiator sequence is single-stranded. In an alternative embodiment, the initiator sequence is double-stranded. It will be understood by persons skilled in the art that a 3’-overhang (/.e., a free 3’-end) allows for efficient addition.

In one embodiment, the initiator sequence is immobilised on a solid support. This allows PolQ and the cleaving agent to be removed (in steps (c) and (e), respectively) without washing away the synthesised nucleic acid. The initiator sequence may be attached to a solid support stable under aqueous conditions so that the method can be easily performed via a flow setup.

In one embodiment, the initiator sequence is immobilised on a solid support via a reversible interacting moiety, such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin orstreptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K.

In a further embodiment, the initiator sequence is immobilised on a solid support via a chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker. Therefore, in one embodiment, the method additionally comprises extracting the resultant nucleic acid by cleaving the chemical linker through the addition of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes for an allyl linker; or TCEP or tris(bipyridine)ruthenium (II) chloride and sodium ascorbate for an azide-masked hemiaminal ether linker.

In one embodiment, the resultant nucleic acid is extracted and amplified by polymerase chain reaction using the nucleic acid bound to the solid support as a template. The initiator sequence could therefore contain an appropriate forward primer sequence and an appropriate reverse primer could be synthesised.

In an alternative embodiment, the immobilised initiator sequence contains at least one restriction site. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by using a restriction enzyme.

The use of restriction enzymes and restriction sites to cut nucleic acids in a specific location is well known in the art. The choice of restriction site and enzyme can depend on the desired properties, for example whether ‘blunt’ or ‘sticky’ ends are required. Examples of restriction enzymes include: Alul, BamHI, EcoRI, EcoRII, EcoRV, Haell, Hgal, Hindlll, Hinfl, Notl, Pstl, Pvull, Sail, Sau3A, Seal, Smal, Taql and Xbal.

In an alternative embodiment, the initiator sequence contains at least one uridine. Treatment with uracil-DNA glycosylase (UDG) generates an abasic site. Treatment on an appropriate substrate with an apurinic/apyrimidinic (AP) site endonuclease will extract the nucleic acid strand.

Nucleic acid synthesis method

In one embodiment, PolQ of the invention is added in the presence of an extension solution comprising one or more buffers (e.g., Tris or cacodylate), one or more salts (e.g., Na+, K+, Mg2+, Mn2+, Cu2+, Zn2+, Co2+, etc., all with appropriate counterions, such as Cl') and inorganic pyrophosphatase (e.g., the Saccharomyces cerevisiae homolog). It will be understood that the choice of buffers and salts depends on the optimal enzyme activity and stability.

The use of an inorganic pyrophosphatase helps to reduce the build-up of pyrophosphate due to nucleotide triphosphate hydrolysis by PolQ. Therefore, the use of an inorganic pyrophosphatase has the advantage of reducing the rate of (1) backwards reaction and (2) polymerase-mediated strand dismutation. Thus, according to a further aspect of the invention, there is provided the use of inorganic pyrophosphatase in a method of nucleic acid synthesis. In one embodiment, the inorganic pyrophosphatase comprises purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae.

In one embodiment, step (b) is performed at a pH range between 5 and 10. Therefore, it will be understood that any buffer with a buffering range of pH 5-10 could be used, for example cacodylate, Tris, HEPES orTricine, in particular cacodylate or Tris.

In one embodiment, step (d) is performed at a temperature less than 99°C, such as less than 95°C, 90°C, 85°C, 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, or 30°C. It will be understood that the optimal temperature will depend on the cleavage agent utilised. The temperature used helps to assist cleavage and disrupt any secondary structures formed during nucleotide addition.

In one embodiment, steps (c) and (e) are performed by applying a wash solution. In one embodiment, the wash solution comprises the same buffers and salts as used in the extension solution described herein. This has the advantage of allowing the wash solution to be collected after step (c) and recycled as extension solution in step (b) when the method steps are repeated.

In one embodiment, the method is performed within a flow instrument, such as a microfluidic or column-based flow instrument (Figure 3). The method described herein can easily be performed in a flow setup which makes the method simple to use. It will be understood that examples of commercially available DNA synthesisers {e.g., MerMade 192E from BioAutomation or H-8 SE from K&amp;A) may be optimised for the required reaction conditions and used to perform the method described herein.

In one embodiment, the method is performed on a plate or microarray setup. For example, nucleotides may be individually addressed through a series of microdispensing nozzles using any applicable jetting technology, including piezo and thermal jets. This highly parallel process may be used to generate hybridization microarrays and is also amenable to DNA fragment assembly through standard molecular biology techniques.

In one embodiment, the method additionally comprises amplifying the resultant nucleic acid. Methods of DNA/RNA amplification are well known in the art. For example, in a further embodiment, the amplification is performed by polymerase chain reaction (PCR). This step has the advantage of being able to extract and amplify the resultant nucleic acid all in one step.

The template-independent nucleic acid synthesis method described herein has the capability to add a nucleic acid sequence of defined composition and length to an initiator sequence. Therefore, it will be understood by persons skilled in the art, that the method described herein may be used as a novel way to introduce adapter sequences to a nucleic acid library.

If the initiator sequence is not one defined sequence, but instead a library of nucleic acid fragments (for example generated by sonication of genomic DNA, or for example messenger RNA) then this method is capable of de novo synthesis of ‘adapter sequences’ on every fragment. The installation of adapter sequences is an integral part of library preparation for next-generation library nucleic acid sequencing methods, as they contain sequence information allowing hybridisation to a flow cell/solid support and hybridisation of a sequencing primer.

Currently used methods include single-stranded ligation, however this technique is limited because ligation efficiency decreases strongly with increasing fragment length.

Consequently, current methods are unable to attach sequences longer than 100 nucleotides in length. Therefore, the method described herein allows for library preparation in an improved fashion to that which is currently possible.

Therefore, in one embodiment, an adapter sequence is added to the initiator sequence. In a further embodiment, the initiator sequence may be a nucleic acid from a library of nucleic acid fragments.

Kits

According to a further aspect of the invention, there is provided the use of a kit in a method of template-independent nucleic acid synthesis, wherein said kit comprises PolQ as defined in the first aspect of the invention optionally in combination with one or more components selected from: an initiator sequence, one or more 3’-blocked nucleotide triphosphates, inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae, and a cleaving agent; further optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

Suitably a kit according to the invention may also contain one or more components selected from the group: an extension solution, a wash solution and/or a cleaving solution as defined herein; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

The following studies and protocols illustrate embodiments of the methods described herein: EXAMPLE 1: Expression of DNA polymerase theta (PolQ) in BL21 DE3 cells

EXAMPLE 4: Example DNA Synthesis method using hPolQ and engineered TdT 1. An immobilised single-stranded DNA initiator is exposed to the extension solution, which is composed of either hPolQ in the case of a 3'-azidomethyl dATP or an engineered TdT in the case of 3'-azidomethyl dTTP, 3'-azidomethyl dCTP, or 3'-azidomethyl dGTP; a 3’-azidomethyl dATP, a 3’-azidomethyl dTTP, a 3’-azidomethyl dCTP, and/or a a 3’-azidomethyl dGTP; inorganic pyrophosphatase (e.g., the Saccharomyces cerevisiae homolog); and required buffers (Tris acetate) and salts (Na+, K+, Mg2+, Mn2+, and/or Co2+, all with appropriate counterions, such as Cl ) and reacted at optimised concentrations, times and temperatures. The 3’-blocked nucleotide triphosphate will contain one of the nitrogenous bases adenine, guanine, cytosine or thymine. 2. The extension mixture is then removed with wash mixture and recycled. Wash mixture is the extension mixture without hPolQ or TdT and the 3’-blocked nucleotide triphosphate. 3. The immobilised (n+1) DNA strand is treated with cleavage mixture composed of an appropriate buffer, denaturant (e.g., urea, guanidinium chloride, formamide, betaine, etc.), and cleavage agent (e.g., tris(bipyridine)ruthenium (II) chloride and sodium ascorbate or tris(2-carboxyethyl)phosphine (TCEP) to de-block a 3’-0-azidomethyl group; palladium complexes to de-block 3’-0-allyl group; or sodium nitrite to de-block the 3’-aminoxy group).

The temperature can be raised up to 99°C to assist in cleavage and disruption of secondary structures. The optimal temperature depends on the cleavage agent utilised. 4. The immobilised deblocked (n+1) DNA strand is treated with wash mixture to remove the cleavage mixture. 5. Cycles 1-4 are repeated with the base-specific 3’-blocked nucleotide triphosphate until the desired oligonucleotide sequence is achieved. 6. Once the desired sequence is achieved, polymerase chain reaction with primers specific to the DNA product is used to directly “extract” and amplify the product.

Claims (29)

1. Use of a DNA polymerase theta (PolQ or ΡοΙΘ) enzyme in a method of template-independent nucleic acid synthesis.

2. The use as defined in claim 1, wherein the PolQ enzyme comprises an amino acid of sequence SEQ ID NO: 1 or a functional equivalent or fragment thereof having at least 20% sequence identity to said amino acid sequence.

3. A method of nucleic acid synthesis, which comprises the steps of: (a) providing an initiator sequence; (b) adding a 3’-blocked nucleotide triphosphate to said initiator sequence in the presence of PolQ as defined in claim 1 or claim 2; (c) removal of all reagents from the initiator sequence; (d) cleaving the blocking group from the 3’-blocked nucleotide triphosphate in the presence of a cleaving agent; (e) removal of the cleaving agent.

4. The method as defined in claim 3, wherein greater than 1 nucleotide is added by repeating steps (b) to (e).

5. The method as defined in claim 3 or claim 4, wherein the 3’-blocked nucleotide triphosphate is blocked by either a 3’-0-azidomethyl, 3’-aminoxy or 3’-0-allyl group.

6. The method as defined in any one of claims 3 to 5, wherein PolQ is added in the presence of an extension solution comprising one or more buffers, such as Tris or cacodylate, one or more salts, and inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae.

7. The method as defined in any one of claims 3 to 6, wherein step (b) is performed at a pH range between 5 and 10.

8. The method as defined in any one of claims 3 to 7, wherein step (b) is alternatively performed with TdT for 3'-blocked dNTP, such as dTTP, dGTP, dCTP.

9. The method as defined in any one of claims 3 to 8, wherein the cleaving agent is a chemical cleaving agent.

10. The method as defined in any one of claims 3 to 9, wherein the cleaving agent is an enzymatic cleaving agent.

11. The method as defined in any one of claims 3 to 10, wherein the cleaving agent is selected from: transition metal complex, such as a photo-activated ruthenium complex, specifically such as tris(bipyridine)ruthenium (II) chloride and sodium ascorbate; reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP); a palladium complex; or sodium nitrite.

12. The method as defined in any one of claims 3 to 11, wherein the cleaving agent is added in the presence of a cleavage solution comprising a denaturant, such as urea, guanidinium chloride, formamide or betaine, and one or more buffers.

13. The method as defined in any one of claims 3 to 12, wherein step (d) is performed at a temperature less than 99°C.

14. The method as defined in any one of claims 3 to 13, wherein steps (c) and (e) are performed by applying a wash solution.

15. The method as defined in any one of claims 3 to 14, wherein the method is performed within a flow instrument, such as a microfluidic or column-based flow instrument.

16. The method as defined in any one of claims 3 to 15, wherein the method is performed within a plate or microarray setup.

17. The method as defined in any one of claims 3 to 16, wherein the initiator sequence is between 5 and 50 nucleotides long, such as between 10 and 30 nucleotides long, in particular approximately 20 nucleotides long.

18. The method as defined in any one of claims 3 to 17, wherein the initiator sequence is single-stranded or double-stranded.

19. The method as defined in any one of claims 3 to 18, wherein the initiator sequence is immobilised on a solid support.

20. The method as defined in claim 19, wherein the initiator sequence is immobilised via a reversible interacting moiety.

21. The method as defined in claim 19, which additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety.

22. The method as defined in claim 21, wherein the reversible interacting moiety is a chemically-cleavable linker, such as a disulfide, allyl or azide-masked hemiaminal ether.

23. The method as defined in claim 21, which additionally comprises extracting the resultant nucleic acid by cleaving the chemically-cleavable linker, such as by the addition of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT) or a palladium complex.

24. The method as defined in any one of claims 3 to 19, wherein the initiator sequence contains at least one restriction site.

25. The method as defined in claim 24, which additionally comprises extracting the resultant nucleic acid by using a restriction enzyme.

26. The method as defined in any one of claims 3 to 25, wherein the initiator sequence contains at least one uridine.

27. The method as defined in any one of claims 3 to 26, which additionally comprises amplifying the resultant nucleic acid, such as by PCR.

28. Use of a kit in a method of template-independent nucleic acid synthesis, wherein said kit comprises a PolQ as defined in any one of claims 1 to 2, optionally in combination with one or more components selected from: an initiator sequence, one or more 3’-blocked nucleotide triphosphates, inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae, and a cleaving agent; further optionally together with instructions for use of the kit in accordance with the method as defined in any one of claims 3 to 27.

29. The use as defined in claim 28, which additionally comprises one or more components selected from the group: an extension solution, a wash solution and/or a cleaving solution.